Immunotherapy for Brain Tumors

In some ways, therapies which stimulate a cancer-fighting immune response are the ideal way to treat the disease. Non-invasive, selective, without toxicity to healthy cells, a properly trained immune system can act as an elite search-and-destroy team.

The bodies of each one of us contains cells in various degrees of pre-malignancy. It is mainly the proper functioning of the immune system which detects these rogue cells and destroys them before they can multiply into an active tumour. The immune system is a complex team composed of many different cell types with various, even opposite, functions. Additionally, immune cells may have differing functions depending on the cytokines and other chemical signals existing in the cellular microenvironment. Due to this plasticity of function, cancer cells have the ability to reprogram, or re-educate tumour infiltrating immune cells such as macrophages, changing their activity from cancer-fighting to cancer-promoting. This is certainly true for brain tumours, particularly high-grade tumours such as glioblastomas.

Natural killer cells have the ability to detect and destroy malignant cells in some types of cancer. There is a common misconception that these cells are also important in the immune response against brain tumours. However, most evidence shows that natural killer cells have a very limited penetration of the blood-brain barrier, and are either rare or absent within the majority of brain tumours. Similarly, one study shows that the presence of T helper and cytotoxic T cells (which can also be trained to destroy cancer cells) is greatly increased in glioblastomas relative to lower grade tumours, and that this increase in T cell infiltration is related to blood-brain barrier leakiness, which occurs with rapid angiogenesis in glioblastoma (1).

Furthermore, the efficacy of immunotherapy may vary according to genetic subtype, even within the same histological grade. For example, in a small trial of dendritic cell vaccine therapy for glioblastoma, the poor prognosis mesenchymal subtype responded very well to the therapy, with a dramatic increase in survival compared to historical controls, while those in the better prognosis proneural subtype did not have any noticeable improvement in survival compared to historical controls (2). This study emphasizes the need for genetic subtyping in order to determine which patients will likely benefit from immunotherapy, and which may not.

A Selection of Completed Trials

Autologous dendritic cells pulsed with tumor lysate

Dendritic cells are immune cells which circulate in the blood, whose main function is to present antigens to other immune effector cells, for the purpose of antigen recognition. Autologous means "from the same patient" and refers to the fact that both the dendritic cells and the tumor lysate which together make the vaccine, are derived from the same patient that receives the finished vaccine.

Dendritic cell based vaccine therapies require sufficient fresh tumour material from the patient receiving the vaccine, as well as the collection of dendritic cells from the patient, by leukapheresis. The dendritic cells are multiplied and co-cultured with the tumour lysate and later the DC cells, now capable of presenting tumour antigens to immune effector cells, are re-injected into the patient’s bloodstream.

A personalized dendritic cell vaccine therapy called DCVax, being developed by Northwest Biotherapeutics, is currently being tested in a phase III trial for glioblastomas, with estimated primary completion date of September 2014.

Belgian researchers conducted a trial of surgical resection followed by dendritic cell therapy (loaded with tumor lysate) for recurrent anaplastic glioma patients, including 18 anaplastic astrocytomas (AA). While only 38% of AA patients were progression-free at six months following vaccination, these same patients were also progression-free at 12 months. Median progression-free survival for the 18 AA patients was 4.6 months (3). While these results are not remarkable, it must be noted that median survival from the time of vaccination was 20.5 months. However, it is unknown to what extent subsequent therapy contributed to this favorable (relative to other trials) survival statistic.

Another form of dendritic cell vaccine utilizes synthetic peptides for glioma-associated antigens, rather than using tumor lysate prepared from the patient’s tumor. Outstanding results for glioblastomas have also been seen using this method.

In a phase I trial (4), the ICT-107 vaccine (ImmunoCellular Therapeutics Ltd.), a dendritic cell vaccine prepared with six synthetic peptides from tumor-associated antigens, was administered to 16 newly diagnosed glioblastoma patients. Progression-free survival of these patients was an impressive 16.9 months, and median overall survival was 38.4 months, comparable to the survival results seen in the phase I DCVax trial. A recent update revealed that 5 year survival was 50% (in other words, half of the patients were still alive at 5 years).

A phase II trial of ICT-107 is underway for newly diagnosed glioblastoma, with estimated primary completion date of October 2014.

On June 1, 2014, ImmunoCellular Therapeutics published a press release in conjunction with their presentation at the 2014 annual meeting of ASCO. This press release included preliminary results of their randomized phase II trial of ICT-107 vaccine for newly diagnosed glioblastoma patients. All patients in the trial underwent surgery and standard radiochemotherapy with temozolomide, and were positive for human leukocyte antigen-A1 or -A2 (HLA-A1, HLA-A2) which are antigen-presenting proteins at the cell surface. 124 patients were randomized to receive either ICT-107 vaccine or placebo. 62% of these patients belonged to the HLA-A2 class.

For the MGMT methylated, HLA-A2 positive group, median survival has not yet been reached in either the treatment or placebo group. Progression-free survival in the vaccinated group is reported to be 24.1 months compared to 8.5 months in the placebo group. These statistics are measured from the time of randomization, after the completion of chemoradiation. The average interval between surgery and randomization in this trial was 83 days (2.7 months). In order to compare these results with other trials measured from the time of diagnosis or trial registration, we must then add approximately 2.7 months onto the reported statistics. A PFS of 24 to 27 months for MGMT methylated patients in the vaccinated group not only surpasses the PFS of the placebo group nearly 3-fold, it also far exceeds the PFS of MGMT methylated patients reported in any other phase II or III trial, to the best of my knowledge.

MGMT unmethylated, HLA-A2 positive patients vaccinated with ICT-107 had a median PFS of 10.5 months, and a median overall survival of 15.8 months, compared to 6 months PFS and 11.8 months OS in the placebo group. Though there is clearly a trend towards improved outcomes in this patient subgroup with vaccination, the differences have not reached statistical significance (PFS p=.442, OS p=.175).

As explained above, these statistics are measured from the time of randomization, after completion of radiochemotherapy. Average interval in this trial between surgery and randomization was 83 days (2.7 months). If we add 2.7 months onto the reported statistics, we can approximate outcomes from the time of surgery/diagnosis. For the vaccinated (and MGMT unmethylated, HLA-A2 positive) group this works out to 13.2 months median PFS and 18.5 months median OS. This would perhaps be the best PFS outcome yet reported for unmethylated MGMT patients in a phase II or III trial for newly diagnosed GBM. A median OS of 18+ months in this subgroup is also one of the best outcomes reported for MGMT unmethylated GBM, surpassed somewhat by the outcome reported in the nimotuzumab trial, described on the Best Trial Results for Newly Diagnosed Glioblastoma page.

A small trial for recurrent malignant gliomas (including 9 anaplastic gliomas) used an innovative variation of the peptide method (5). In this trial, special culture methods for the dendritic cells were used to induce alpha-type 1 polarized DCs (aDC1) which are more effective than standard mature dendritic cells. Additionally, patients also received poly-ICLC intramuscular injections to further boost the immune response. The median progression-free survival of the 9 anaplastic gliomas patients (5 astrocytoma, 3 oligodendroglioma, 1 oligoastrocytoma) was 13 months, which is one of the best median PFS cited in the literature for recurrent anaplastic gliomas.

Table of currently recruiting vaccine and other immunotherapy trials for adult glioma

Improving the Efficacy of Immunotherapy

COX-2 inhibitors

Parecoxib plus glioma cell immunization in vivo

A study (8) published in Journal of Neuroimmunology (July 2014, online) shows that the addition of a selective COX-2 inhibitor to immunization with irradiated whole glioma cells is curative for rats implanted intracranially with syngeneic rat glioma cells. Monotherapy with either irradiated glioma cell immunization, or COX-2 inhibition alone were not curative.

Fischer 344 rats were first implanted with the syngeneic N32 rat glioma cells. On days 1, 15, and 29, the rats were immunized subcutaneously with irradiated (80 Gy) N32 cells into the right thigh, in the attempt to arouse an immune response against the grafted brain tumours. Additionally, parecoxib (a selective COX-2 inhibitor) was pumped into two groups of rats intraperitoneally for either the first 28 days, or on days 7-13 and 17-23.

According to Figure 1, all untreated tumour-bearing rats died before day 30. Parecoxib-treated rats did not have improved survival compared to untreated controls. Rats immunized with irradiated glioma cells had a statistically significant improvement in survival, with several rats surviving to around day 40.

Dramatically, the combination of the COX-2 inhibitor parecoxib and immunization led to long-term survival (beyond day 160) and apparent cures in the majority of rats. The groups of rats treated with continuous parecoxib (day 1-28) plus immunization had a 60% cure rate, while the rats receiving intermittent parecoxib plus immunization had a 50% cure rate.

At day 170, the surviving rats were challenged again with tumour cells in the opposite brain hemisphere, but given no further treatment. 89% of the surviving rats that had previously received immunotherapy plus continuous parecoxib survived the tumour re-challenge (to at least day 270), while 43% of the rats that had previously received immunotherapy plus intermittent parecoxib survived the tumour-rechallenge.

When the blood of the various groups of rats was analyzed, the combination-treated rats were found to have much increased levels of interferon-gamma (one of the key cytokines involved in type 1 immunity) as well as increased numbers of CD8+ effector memory T cells. Rat brains from tumour re-challenged (and previously combination-treated) rats were shown to have massively increased infiltration of TCR (T cell-receptor) and CD8 positive cells, mediating the anti-tumour response.

Parecoxib is an injectable COX-2 inhibitor available in Europe as a treatment for post-operative pain. The drug was refused approval in 2005 by the FDA. Perhaps the most commonly prescribed selective COX-2 inhibitor is the orally administered celecoxib (Celebrex). Several clinical trials and retrospective studies have looked at Celebrex as an addition to standard therapies for malignant glioma patients, though the outcomes were inconclusive. As the COX-2 enzyme and its downstream products, especially prostaglandin E2 (PGE2) play a large role in cancer-induced immunosuppression, it is likely that COX-2 inhibitors such as Celebrex will be found most useful as an addition to cancer immunotherapy, such as dendritic cell vaccines.

Celecoxib plus tumor-lysate pulsed dendritic cells in vivo

In this study (9) published in 2013, Wistar rats were implanted intracranially with C6 rat glioma cells, followed by one of the following treatments: a) untreated. b) unpulsed dendritic cells. c) C6 glioma lysate pulsed dendritic cells. d) celecoxib (Celebrex) fed in the diet. e) celecoxib plus C6 lysate pulsed dendritic cells. Dendritic cells were injected subcutaneously on days 3, 10, and 17, while celecoxib was given daily in the diet.

Median survival in the untreated control group was 21 days. Nonpulsed dendritic cell treatment prolonged median survival to 30 days. C6 pulsed dendritic cell vaccination alone, or celecoxib treatment alone both prolonged median survival further to around 40 days. Finally, the combination of celecoxib plus C6 pulsed dendritic cells prolonged median survival to around 55 days, or a 2.6-fold increase in survival time versus untreated controls. In this last group, half the rats were still alive at 60 days when the experiment was ended.

The combination treatment also had the greatest effect in reducing tumour blood vessel density, increasing apoptosis, reducing immunosuppressive cytokines such as IL-10 and PGE2, increasing the type 1 immune cytokine IL-12, and increasing the cytotoxic activity of rat splenocytes.

Notably, the dose of celecoxib (Celebrex) orally fed to the rats in this study, when converted to a human equivalent dose, is within the therapeutic dose range prescribed to humans (200 mg per day, 400 mg per day max).

Imiquimod (Aldara) cream

Imiquimod (trade name Aldara) is an immune response modifier which acts through toll-like receptor 7 on immune cells. Imiquimod is FDA approved for skin conditions such as actinic keratosis, external genital warts, and as of 2004, superficial basal cell carcinoma (a type of skin cancer). At least two trials testing dendritic cell vaccines for glioma patients are adding imiquimod or the related investigational drug resiquimod to the vaccine therapy as adjuvants (see NCT01792505 and NCT01204684).

Unexpectedly, a preclinical study (10) with mice conducted at the University of Minnesota found that imiquimod as a single agent inhibits the growth of intracranial gliomas in the syngeneic GL261 mouse model. In this experiment, mice were divided into 4 groups after implantation of GL261 tumours and received treatment with either a) GL261 cell lysate as immunization to stimulate an immune response b) GL261 cell lysate injected into an Aldara topical application site c) Aldara alone d) saline control. Though the mice given Aldara topical application alone were intended as a control group, their median survival was increased by 50% relative to the saline only group (from 28 days to 42 days). By day 21, tumour burden was reduced 3-fold in the Aldara alone group relative to the saline control group.

Further experiments were conducted to determine Aldara's mechanism in the treated mice. GL261 tumour-bearing mice were treated every 7 days with topical Aldara cream and immune cells were analyzed on day 22. Aldara treatment depleted T-cell and B-cell populations in the blood with an increase in circulating natural killer and dendritic cells. In contrast, in the cervical lymph nodes (in the neck, which are the draining lymph nodes of the brain) Aldara significantly increased the absolute number and percentage of CD4 T-cells, CD8 T-cells and dendritic cells.

The effect of Aldara was then investigated in brain infiltrating leukocytes at the tumour site. Aldara did not effect B-cell or natural killer cell infiltration. Remarkably, treatment with Aldara nearly doubled the number of dendritic cells and CD4 T-cells in the brain, tripled the number of CD8 (cytotoxic) T-cells, and quadrupled the number of CD8 T cells which displayed CD107a cell surface mobilization in response to GL261 antigen. CD107a is a marker of lytic granules, which cytotoxic CD8 T-cells use to destroy their targets. Immunosuppressive Tregs were depleted in the brain by Aldara treatment.

In further testing, brain-infiltrating leukocytes from saline treated mice did not produce interferon-gamma, while BILs from Aldara treated mice produced interferon-gamma in a GL261 antigen-dependent manner.

Caveats: the ability of imiquimod cream (Aldara) to reproduce these impressive effects in humans is unknown. The authors of this study note that rodents and humans have differing thickness of skin and differing subsets of cells which express toll-like receptor 7. Also, the relative surface area of the skin to which Aldara was applied in the study was larger than commonly applied to humans. The authors conclude that further study with Aldara as a single agent in human cancer trials is warranted and also suggest testing low-dose systemic application of imiquimod or similar drugs (as opposed to topical).

Poly-ICLC

Poly-ICLC is a stabilized double-stranded RNA viral mimic, which stimulates the innate and adaptive immune response. In glioma trials, it is injected into muscle tissue. While a small preliminary trial (6) with Poly-ICLC showed impressive results for 11 anaplastic astrocytoma patients (most of whom were also treated with CCNU chemotherapy), a larger more recent trial of Poly-ICLC for 45 recurrent anaplastic gliomas did not replicate that success (7). In the larger trial, 51% of patients had response or stable disease following Poly-ICLC monotherapy, while only 24% were still progression-free at six months. The greater promise of Poly-ICLC is likely as an addition to dendritic cell vaccine therapies.

Metronomic chemotherapy is the use of low daily doses of standard chemotherapy agents. This is in contrast with maximum tolerated dosing strategies in which short bursts of high-dose chemotherapy are followed by a recovery period, due to the toxicity of these high-dose regimens. There are several rationales for metronomic chemotherapy, including anti-angiogenic effects and surprisingly, anti-tumour immunological effects. In this section I will discuss the immunological effects of low dose and intermittent chemotherapy.

In one of the most striking studies (11) of the immunological effects of ultra-low dose temozolomide, approximately 70% of mice survived long term (over 100 days) when given a combination of daily low dose TMZ (2.5 mg per kilogram mouse body weight) with a tumor lysate-pulsed dendritic cell vaccine. In contrast, no mice survived to 100 days when given low-dose TMZ alone, and less than 20% survived long term when given the dendritic cell vaccine alone. This model consisted of intracranial implantation of syngeneic GL26 mouse glioma cells, intraperitoneal delivery of TMZ on days 2-6 after tumour implantation, and subcutaneous DC vaccinations on days 4, 11 and 18.

To investigate the mechanisms behind the increased efficacy of combined dendritic cell vaccination and low-dose temozolomide, splenocytes were harvested from some of the mice in each group on day 35. At a 40:1 immune effector cell to tumour cell ratio, the immune cells from mice treated with either TMZ alone, dendritic cell vaccination alone, or TMZ plus unaltered dendritic cells all had a tumour cell killing efficacy (specific lysis) of approximately 30%. Immune cells from mice treated with combined TMZ and lysate-pulsed dendritic cells had a killing efficacy of nearly 50% and this cell killing was specific to the target GL26 glioma cells. In further testing, CD4+ and CD8+ T-cells from mice treated with both TMZ monotherapy and lysate-pulsed dendritic cell vaccine monotherapy had increased expression of interferon-gamma (a cytokine stimulating type 1 immune responses), with maximum expression of interferon-gamma in the combined TMZ and dendritic cell vaccinated mice. Furthermore, increased infiltration of CD4+ and CD8+ T-cells were observed in the brains of combination treated mice versus mice treated with either TMZ or lysate-pulsed dendritic cells alone.

In vitro, high concentrations of TMZ (100 to 400 micromolar) led to surface expression of calreticulin on the GL26 glioma cells at 48 hours, a sign of immunogenic cell death. In a final experiment, splenocytes from tumour-bearing mice treated and not treated with TMZ were examined at various time points and the frequency of immunosuppressive regulatory T-cells (Tregs) was determined. By days 22, 29 and 36, the TMZ-treated mice had suppressed Treg frequency (similar to non tumour-bearing mice), while the untreated tumour-bearing mice showed a marked increase in the frequency of immunosuppressive Tregs.

In summary, very low dose daily temozolomide (a mouse dose which would be allometrically scaled to a human dose of approximately 10 mg per square meter of body surface) seems to increase the tumour killing efficacy of T-cells and infiltration of those T-cells into mouse brain tumours, which may be directly related to the depletion of immunosuppressive Tregs by the low-dose TMZ treatment. High concentrations of TMZ in vitro led to immunogenic cell death of GL26 mouse glioma cells, though this was not demonstrated in vivo in this study. Of note, a prior study (12) had also shown the depletion of Tregs in rats bearing RG2 rat gliomas with even lower doses of temozolomide (0.5 mg per kilogram rat body weight). However, survival was slightly but not significantly prolonged in this study by the low-dose TMZ treatment, indicating that the very low-dose metronomic TMZ schedule may work best in combination with other immunotherapies such as dendritic cell vaccines.